Original Research Paper
Slov Vet Res 2008; 45 (4): 121-6
UDC 636.3.09:616.336-078:616.15:612.32:577.175.73
USE OF PLASMA GASTRIN AND PEPSINOGEN LEVELS AS
DIAGNOSTIC MARKERS OF ABOMASAL DYSFUNCTION IN
MARWARI SHEEP OF ARID TRACT
Nalini Kataria1*, Anil Kumar Kataria2, Ajey Kumar Gahlot3
1
Department of Veterinary Physiology, 2Apex Centre for Animal Disease Investigation, Monitoring and Surveillance, 3Department of Veterinary Medicine, College of Veterinary and Animal Sciences, Bikaner- 334 001, Rajasthan, India
*Corresponding author, E-mail: [email protected]
Summary: Hormone gastrin is secreted from gastrin cells of pyloric region of abomasum into the blood circulation, thence
to reach the parietal cells and is an important stimulator of acid and pepsinogen secretion. Pepsinogen, a proenzyme is
an inactive form of pepsin which is the most important proteolytic enzyme of gastric juice. To assess the role of gastrin and
pepsinogen in the diagnosis of abomasal parasitism or disorders, the gastrin and pepsinogen levels were determined in
the plasma of Marwari breed of sheep belonging to farmers’ stock of arid tract of Rajasthan state, India. The animals, from
which the blood samples collected, were grouped into healthy, haemonchus infected and drought affected. In healthy animals sampling was carried as one time random sampling and three times sampling. The overall mean values of plasma gastrin and pepsinogen in healthy Marwari sheep were 103.45± 10. 41 pg/ml and 153.61± 13.21 mU tyrosine, respectively.
In haemonchus infected and drought affected sheep a significant (p≤0.05) increase was observed in the mean values for
both the parameters in comparison to that of healthy stock. The highest values for both the parameters were observed in
haemonchus infected animals. The sampling time did not affect the gastrin and pepsinogen levels. This showed that feeding did not affect the levels of gastrin and pepsinogen. The responses of gastrin and pepsinogen in affected sheep indicated that they can be used as diagnostic markers in animals affected with abomasal dysfunctions. Probably the presence
of parasites or inanimate objects in pica damaged the mucosa causing an impairment of abomasal function. Damage to mucosa is related with higher gastrin and pepsinogen release. Further the results suggested that one time random sampling
can be carried out in suspected clinical cases for the determination of plasma gastrin and pepsinogen. The data obtained
can be used as a base line for the future studies in this direction in Marwari sheep or other breeds of sheep.
Key words: drought; gastrin; haemonchosis; Marwari sheep; pepsinogen; pica
Introduction
Hormone gastrin is secreted from gastrin cells
of pyloric region of abomasum into the blood circulation, thence to reach the parietal cells and is an
important stimulator of acid and pepsinogen secretion (1). Pepsinogen, a proenzyme is an inactive form
of pepsin which is the most important proteolytic
enzyme of gastric juice. In the gastric or abomasal
lumen pepsinogen is converted into pepsin in the
Received: 30 April 2008
Accepted for publication: 17 October 2008
presence of acid (1, 2). In ruminant blood a certain
physiological level of pepsinogen exists (3). Blood
levels of pepsinogen can be used in the diagnosis of
abomasal parasitism or disorders (4). The increased
plasma levels of pepsinogen are caused due to its
leakage into blood vessels from damaged abomasal
mucosa (5). Increased activation of pepsinogen into
pepsin by enhanced acidity of gastric contents can
cause ulcers in humans and animals (6).
There are reports, although scant, on the use of
plasma pepsinogen and gastrin as indicators of gastric dysfunctions in man (7, 8) and animals (4, 9, 10).
However, there is no literature available on this as-
122
N. Kataria, A.K. Kataria, A.K. Gahlot
pect in Marwari breed of sheep. Also, there is rarity
of literature regarding normal values of pepsinogen
and gastrin in animals.
The Marwari sheep is an important breed of
sheep in arid regions and frequently face problems
of droughts (11). During drought periods animals
feed on non-conventional feeds and develop pica
in which condition they start eating in-animate
objects (2).The gastrointestinal worm infestation
is also a great problem in sheep. In these circumstances mucosa of abomasum is damaged greatly
impairing digestive functions as proper gastric mucosa plays an important role in digestive processes.
However, many a times damage to abomasal mucosa remains undiagnosed. The determination of
plasma levels of gastrin and pepsinogen can be an
important aid for taking timely measures to treat
such animals (2, 5, 6). The present investigation
was planned to explore the possibilities of role of
plasma gastrin and pepsingen as diagnostic tools
in assessing abomasal involvement in Marwari
sheep and to set the normal values of these parameters. In the field conditions to overcome the difficulty of repeated sampling, the investigation was
also aimed to collect samples at different times to
find out the effect, if any, of different sampling time
on the levels of gastrin and pepsinogen.
Material and methods
Plasma gastrin and pepsinogen levels were determined in sheep of Marwari breed belonging to farmers’ stock of arid tract of Rajasthan state, India. The
animals were maintained on the free-range grazing
system. The animals, from which the blood samples
collected, were grouped into healthy, haemonchus
infected and drought affected. In healthy animals
sampling was carried out in two patterns i.e. one
time random sampling and three times sampling.
In one time random sampling cases, the blood was
drawn once, irrespective of time, for the preparation
of plasma from 17 apparently healthy adult Marwari sheep of either sex (10 male and 7 female). The
samples were collected as a part of routine health
checkup during moderate ambience (maximum
temperature varied between 26 and 29 °C) and animals were free from endo-parasites as assessed by
routine faecal examination. In three times sampling
cases blood was obtained from the same 17 adult
Marwari sheep by samplings three times i.e. morning, mid-day and evening and the means were presented irrespective of sex.
In the haemonchus infected group, blood samples
were obtained (from the slaughter house) from other
18 Marwari sheep of either sex (9 male and 9 female)
at the time of slaughtering which had haemonchus
infection detected at the time of slaughter.
In the drought affected group, 20 blood samples
(10 male and 10 female) were collected from drought
affected Marwari sheep of arid tract having the history of pica belonging to farmers’ stock.
All the samples were collected in sterile tubes
containing EDTA (di-potassium) as an anticoagulant for plasma separation. Plasma gastrin levels
were determined by double antibody gastrin 125I
radioimmunoassay (RIA Kit, DPC, USA) as per the
manual supplied with RIA kit in the Radio Isotope
Laboratory, College of Veterinary and Animal Science, Bikaner, India. The materials supplied in the
kit included gastrin antiserum, 125I gastrin, gastrin
calibrators and precipitating solution consisting of
goat anti-rabbit gamma globulin and dilute polyethylene glycol in saline. In the double antibody gastrin
procedure a competition was there between 125I labeled gastrin and gastrin hormone in plasma for the
sites on gastrin specific antibody. After incubation
for 2 hours (15-28oC), bound was separated from free
by PEG accelerated double antibody method. A foam
decantation rack was used to decant the supernatant, retaining the precipitate for counting. Counting was carried out by using 125I Gamma counter
(EC, India).
Plasma pepsinogen was determined by a colorimetric method (7) with modifications (12) using
bovine albumin fraction V (CDH, India) and glycine
(Loba Chemie, India) to prepare substrate mixture.
In the method plasma was added to a substrate
mixture (0.9 % bovine albumin fraction V in glycine
buffer, pH 2.0) and mixed thoroughly. An aliquot of
this mixture was incubated in a water bath at 37oC
for 24 hours. Then reaction was stopped by adding 4
% trichloroacetic acid (Glaxo, India) solution (12).After filtration of the protein precipitate the concentration of acid soluble tyrosine was determined by Folin
Coicalteau’s reagent (CDH, India). For this in each 2
ml of filtrate 0.5 ml of Folin Coicalteau’s reagent and
10 ml of 0.25N sodium hydroxide (Qualigens, India)
were added. Two ml of tyrosine (BDH, India) standard
was also treated in the same way. The optical density
was read at 680 mµ wave length using a spectrophotometer (Systronics, India). The enzyme activity was
expressed as milli-units (mU) tyrosine (13).
Correlation was determined by MSTAT computer
programme. Statistical significance for individual
Use of plasma gastrin and pepsinogen levels as diagnostic markers of abomasal dysfunction in Marwari sheep of arid tract
parameter between male and female, healthy and
infected, healthy and drought affected, morning and
mid-day sampling, and morning and evening sampling was analysed by paired- t test (14).
Results
The mean values of plasma gastrin and pepsinogen in Marwari sheep are presented in two tables.
Mean values that are presented in the table 1 belong to healthy, haemonchus infected and drought
affected animals. They are further grouped into
male and female animals. Non significant (p>0.05)
difference was observed in the respective mean
values of plasma gastrin and pepsinogen between
male and female within each category i.e. healthy,
haemonchus infected and drought affected animals.
Table 1: Plasma levels of gastrin and pepsinogen in
healthy, haemonchus infected and drought affected Marwari sheep
S.No.
Groups
1.
(a)
(b)
(c)
2.
(a)
(b)
(c)
3.
(a)
(b)
(c)
Healthy
Overall (17)
Male (10)
Female (7)
Haemonchus Infected
Overall (18)
Male (9)
Female (9)
Drought affected
Overall (20)
Male (10)
Female (10)
Gastrin
( pg/ml)
Pepsinogen
(mU tyrosine)
103.45± 10.41
100.32±11.41
106.41± 13.21
153.61± 13.21
150.41± 14.11
156.4± 11.61
489.61 ±12.33 bd
485.45 ±13.6 bd
494.32 ±11.00bd
772.31± 12.2 bd
769.21 ±10.12 bd
776.30 ±14.40 bd
310.33± 15.1 bd
308.97± 16.0 bd
311.51± 14.9 bd
460.01± 11.5 bd
457.87± 12.0 bd
363.45± 10.3 bd
1. Figures in the parentheses indicate number
of animals.
2. Healthy overall mean value of each parameter was compared with the respective mean value
of infected and drought affected animals. The significant (p≤0.05) variation has been shown by superscript ‘b’.
3. The mean values of each parameter of
haemonchus affected and drought affected animals were compared and means superscribed by
letter‘d’ within a given parameter differed significantly (p≤0.05) from the respective means of other
group.
123
The overall mean values of plasma gastrin and
pepsinogen of haemonchus infected and drought
affected animals were significantly (p≤0.05) higher
than the respective overall mean value of healthy
animals. Haemonchus infected animals showed a
greater rise in the mean values of both the parameters as compared to drought affected animals.
Table 2: Plasma levels of gastrin and pepsinogen in healthy
Marwari sheep
S.No. Groups
Gastrin
(pg/ml)
Pepsinogen
(mU tyrosine)
A.
One time random
sampling Overall (17)
103.45± 10.41
153.61± 13.21
B.
Three times sampling
Overall, three times
sampling (51)
Morning sampling (17)
Mid –day sampling (17)
Evening sampling (17)
104.0± 10.12 a
99.2± 11.0 ac
105.0± 9.23 ac
109.8± 11.3 ac
152.6 ± 13.0 a
147.5±12.75 ac
152.7 ± 15.62 ac
159.4± 12.56 ac
(a)
(b)
(c)
1. Figures in the parentheses indicate number
of observations from 17 healthy animals.
2. Overall mean value of one time random
sampling of each parameter was compared with the
respective mean value of overall three times sampling, morning, mid-day and evening sampling. The
non-significant (p>0.05) variation has been shown
by superscript ‘a’.
3. The non-significant (p>0.05) variation
among the mean values during morning, mid-day
and evening samplings has been shown by superscript ‘c’.
Mean values presented in the table 2 are of
healthy animals categorised according to one time
random sampling and three times sampling. Overall
mean value of one time random sampling of each
parameter was compared with the respective mean
value of overall three times sampling, morning,
mid-day and evening sampling. The non-significant
(p>0.05) variation was observed in each case. Further in three time sampling category the mean values obtained during morning, mid-day and evening
samplings were compared from each other and the
variations were non-significant (p>0.05) among the
mean values.
A significant (p≤0.05) correlation (г =0.921*) between the values of plasma gastrin and pepsinogen
in haemonchus infected and drought affected sheep
was observed.
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N. Kataria, A.K. Kataria, A.K. Gahlot
Discussion
The overall mean value of plasma gastrin in the
present study for healthy animals was found in the
middle range of the values obtained by earlier workers in sheep (15,16) and it was more or less similar
to those recorded in calves (9) and cows (17). The
healthy overall mean value of plasma pepsinogen
in the present study was almost comparable to
those given for lambs (4) and sheep (16).
The non significant (p>0.05) effect of sampling
time showed that feeding did not affect the levels of
gastrin and pepsinogen in present study. It could
be due to the fact that in ruminants the abomasum
receives a continuous, though variable inflow of
forestomach material (18) therefore sampling time
did not affect the values of gastrin and pepsinogen.
Earlier report (15) also suggests that mean plasma
gastrin concentrations do not increase in response
to feeding.
The pattern of changes in the values of plasma
gastrin and pepsinogen was similar in the present
study. The values of both the parameters increased
in haemonchus infected and drought affected animals when compared to healthy animals. Similar
findings were reported in camel (2). Serum gastrin
concentrations were observed elevated in parasitised sheep (19). Earlier researchers (4) found an increase in plasma gastrin and pepsinogen levels in
lambs infected with haemonchiasis and in sheep
infected with Ostertagia circumcincta (6, 16).
Earlier workers have reported increased levels
of serum pepsinogen in cattle with abomasal ulcers (6, 20); of plasma gastrin in cattle with bleeding abomasal ulcers (17) and of serum pepsinogen
and gastrin in bovine ostertagiasis (9, 12).
A significant correlation between the values of
plasma gastrin and pepsinogen in haemonchus infected and drought affected sheep (4) indicated that
both the parameters can be used simultaneously
or individually to assess the abomasal dysfunction.
Plasma pepsinogen estimations are performed routinely in many laboratories as an aid in the diagnosis of ostertagiasis. Elevated values also occur
in haemonchosis and levels decline after effective
anthelmintic treatment. Plasma gastrin concentrations reflect the size of abomasal worm burden in
both young and older animals (21).
Haemonchus contortus is one of the most abundant infectious agents in sheep around the world,
causing great economic damage to sheep farms.
Nematode larvae developing within the glands
cause local loss of parietal cells and mucous cell
hyperplasia whereas reduced hydrochloric acid
secretion, increased serum gastrin and pepsinogen concentrations and generalized histological
changes are associated with parasites in the abomasal lumen. Parietal cells with dilated canaliculi
and degenerative changes typical of necrosis are
present soon after the transplantation of adult
worms, and abomasal secretion is also affected
(22). The massive doses of parasites produce a
significant decrease in acidity and increase in sodium ion concentration of abomasal fluid. This
is followed by an increase in plasma pepsinogen
(23). The changes in abomasal environment are
due to increased permeability of mucosa. Rise in
abomasal pH is an important stimulus for gastrin
release (2).
Anaerobic bacteria survive in greater numbers
as the pH rises. Failure to lyse bacteria may affect
adversely the nutrition of the host. The parasites
may initiate the pathophysiology through the release of excretory or secretory products which
either act directly on parietal cells or indirectly
through enterochromaffin-like cells by provoking inflammation or by disrupting the protective
mucosal defense system. Parietal cell dysfunction
leads to loss of mature chief cells, mucous cell hyperplasia and hypergastrinaemia. Inflammation
increases circulating pepsinogen concentrations
and may also contribute to increased gastrin secretion (22).
The responses of gastrin and pepsinogen in affected Marwari sheep in the present study certainly indicated towards their diagnostic significance
in abomasal dysfunctions. Probably the presence
of parasites or inanimate objects in pica damaged
the mucosa causing an impairment of abomasal
function. Recent work in camel has demonstrated
a marked increase in levels of the plasma gastrin
and pepsinogen in pica affected camels which had
sand in their third compartment damaging the mucosa (2). Damage to mucosa causes a drop in hydrochloric acid production (22) resulting in elevation of gastric pH which is accompanied by higher
gastrin and pepsinogen release (16, 22, 24). Further the results suggested that one time random
sampling can be carried out in suspected clinical
cases for the determination of plasma gastrin and
pepsinogen as reported by earlier workers (2). The
data obtained can be used as a base line for the
future studies in this direction in Marwari sheep or
other breeds of sheep.
Use of plasma gastrin and pepsinogen levels as diagnostic markers of abomasal dysfunction in Marwari sheep of arid tract
Acknowledgements
Authors are thankful to Dean, College of Veterinary and Animal Science, Bikaner for providing necessary facilities to carry out this work.
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126
N. Kataria, A.K. Kataria, A.K. Gahlot
UPORABA VREDNOSTI GASTRINA IN PEPSINOGENA V KRVNI PLAZMI ZA DIAGNOSTIČNE
POKAZATELJE OKVARE SIRIŠČNIKA PRI OVCAH PASME MARVARI V SUŠNIH PODROČJIH
N. Kataria, A.K. Kataria, A.K. Gahlot
Povzetek: Hormon gastrin izločajo gastrinske celice v piloričnem delu siriščnika v krvni obtok. Preko krvi gastrin prihaja
do parietalnih celic in pomembno vpliva na izločanje želodčne kisline in pepsinogena, ki je proencim, neaktivna oblika
pepsina, najpomembnejšega proteolitičnega encima v želodčnem soku. Da bi določili vlogo gastrina in pepsinogena pri
diagnostiki zajedavskih invazij in drugih motenj siriščnika, smo določili vrednosti gastrina in pepsinogena v krvni plazmi ovc
pasme marvari, ki so del čred v polpuščavskem delu zvezne države Radžastan v Indiji. Živali, pri katerih je bila odvzeta kri,
smo razdelili v tri skupine: zdravo, invadirano s hemonhusi in prizadeto zaradi pomanjkanja vode. Pri zdravih živalih smo
vzorce krvi jemali na dva načina: z enkratnim naključnim odvzemom ali pa trikrat zapored v istem dnevu. Skupne povprečne
vrednosti gastrina in pepsinogena v plazmi zdravih ovc so bile 103,45 ± 10,41 pg/ml oz. 153,61 ± 13,21 mU tirozina. Pri
obeh skupinah, prizadetih bodisi zaradi zajedavcev ali suše, smo opazili statistično značilno (p £ 0,05) povečanje obeh
parametrov v primerjavi z zdravimi živalmi. Čas jemanja krvi ni vplival na vrednosti gastrina in pepsinogena, kar pomeni, da
tudi hranjenje ne vpliva na njune vrednosti. Ker pa se oba parametra spremenita pri prizadetih živalih, ju lahko uporabimo za
diagnostična pokazatelja okvare siriščnika. Verjetno tako zajedavci kot neorganski predmeti, ki jih ovce zaužijejo med bolezenskim poželenjem po nenaravnih hrani, poškodujejo želodčno sluznico in s tem okvarijo delovanje siriščnika. Poškodba
sluznice ima za posledico tudi povečano sproščanje gastrina in pepsinogena. Rezultati preiskave so tudi pokazali, da ob
sumu, da gre za klinične primere, zadošča enkratni odvzem krvi ne glede na čas dneva oz. hranjenja. Pridobljeni podatki
lahko služijo za osnovo pri nadaljnjih proučevanjih obremenitve ovc marvari in drugih pasem.
Ključne besede: suša; gastrin; haemonchosis; ovce marvari; pepsinogen; uživanje nenaravne hrane